Tophat2 with fusion search and tophat-fusion-post problems

[seqfast]

Updated:

You must not rename any files in the tophat output directories. My scripts do this automatically, renaming the output files with the sample names - this will cause tophat-fusion-post to fail.


Hi all,

First off thanks to the developers for what is an already excellent package. Tophat2 works great for me and I look forward to using it more.

On to my question - has anyone successfully used tophat-fusion-post with Tophat2 --bowtie1 using the fusion search options?

I am running Linux, using the pre-built Linux binaries.

Tophat2 with --bowtie1 completes fine, I have a large fusions.out file. For my current datasets I have between 350k and 1M candidates - filtering is absolutely necessary, and I've not seen that simply screening for high coverage numbers will help (repeats, narrow coverage windows, etc - all the reasons they list in the paper for having the further filtering steps).

I've made the structure as indicated on the Tophat/Fusion webpage, with tophat_* directories with all the typical output files in the directory, the ensGene, refGene and mcl files.

I have two copies of tophat-fusion-post, one from the original Tophat-Fusion, and one from Tophat2. They are not identical. Both behave similarly.

My Tophat2 directory does not have the annotations (ensGene.txt etc.) but my Tophat-Fusion directory does - I have used those, though tophat-fusion-post from Tophat-Fusion wants refGene_sorted.txt, whereas the Tophat2 version does not.

For testing purposes, I have used skip-blast. tophatfusion_out/ is created but fusion_seq.fa is empty. bowtie does try to align, but nothing happens. Everything completes with no errors, just 0 fusions found.

As a test, I also ran this in a completely empty directory, and it does not complain. This is worrisome, since the program should complain it has no files to work with! Based on this, I think it might not be properly reading the fusions.out file to begin with, even though it's in the directory in the structure on their webpage.

Tophat-fusion is in my path, and bowtie, bowtie2 (not that i'm using it), tophat and tophat2 all call the proper programs. All the bam files have "chr" as a prefix, as do the ensGene and refGen files.

Hopefully someone has used Tophat2 with --bowtie1 and then used tophat-fusion-post on the resultant fusions.out and can provide some advice.

Thank you all for your time,

-sf

[bharati]

Ya i tried Tophat2 with --bowtie1 and then used tophat-fusion-post on the resultant fusions.out but got same result as told by you ( 0 filtered fusions, with no errors). Dont know how to deal with it.

Anybody please come up with an answer.

[Charitra]

Bharati
try reading my message in the post " tophat-fusion-post result empty" for the detailed information. I solved the problem..

[NikTuzov]

Hello:

Could you please help me with a basic question.

The documentation on this page:

http://tophat.cbcb.umd.edu/fusion_manual.html#output

says nothing about where to find the fusion score.
I suspect it's the value 0.569598 in the file fusions.out.
If I am wrong, where do I find the score?

Regards,
Nik

[Charitra]

please open html file or fusion output file with excel then you can find it.
do reply if I have misunderstood your question...

[Charitra]

DeFuse vs Tophat-fusion RNA-seq

does anybody compared RNA-seq fusions using Tophat2 vs DeFuse?
My results from Tophat2 Fusions doesnt match with DeFuse...entirely different ..???
Which one is to choose for validation ?
How do we select for Fusion Gene Validation using wet lab. ?

[NikTuzov]

deFuse and TopHat-Fusion are used together here:

Transcriptome Profiling of the Cancer, Adjacent Non-
Tumor and Distant Normal Tissues from a Colorectal
Cancer Patient by Deep Sequencing
Yan’an Wu1*, Xuetao Wang1, Fangbo Wu1, Ruolei Huang2, Fangqin Xue2, Guantao Liang1, Min Tao1,
Pengwei Cai1, Yi Huang1

They also got very different results, which didn't prevent them from publishing.

[Charitra]

Thank you NikTuzov
you really gave a great support providing yan'an et al. reference...
However, can somebody tell me a good reference book or guide to learn Fusion details like which one is to select for validation from fusion results... some deep understanding...

Thank you in advance...

[NikTuzov]

Are you saying that the score is not available unless I run tophat-fusion-post option? I hoped to find it in fusions.out produced by --fusion-search option.

[Charitra]

Hi
The the scores are available there but fusion-post filters the total fusions.
I have a question about tophat insert size commands, say insertsize 100~380(main 150); how can we modify in tophat commands ?
thank you

]

Are you saying that the score is not available unless I run tophat-fusion-post option? I hoped to find it in fusions.out produced by --fusion-search option.